Ras is known to modulate many biological processes involved in the regulation of cell proliferation, differentiation, development, survival, and motility. Mutations in Ras can lead to a variety of diseases, including cancer. The ERK-MAP kinase cascade plays a major role in Ras-regulated signaling. In this pathway, the ERK and RSK family of protein kinases are key players in signal transmission. Although the regulation of ERKs and their targets have been studied extensively, how RSKs mediate the biological functions of Ras is poorly understood. Our goal is to investigate and define RSK function by characterizing its complex regulation and its downstream targets. During the last grant period we began characterizing the molecular basis of RSK1 activation and some of RSK1's biological functions. We defined a role for RSK1 in cell survival signaling with the discovery of the proapoptotic protein BAD as a RSK substrate. We also continued our characterization of another RSK target, c-Fos, and its role in cell proliferation and oncogenesis. In the continuation of our work on RSK, we propose three major objectives. The first objective of the proposed research is to define at a molecular level how RSK is regulated by multiple protein kinase inputs and site-specific phosphorylation. Given its important role in Ras signaling and the fact that understanding RSK activation would provide the foundation for understanding the regulation of many similar protein kinases, this is an important aim requiring extensive analyses. The second objective is to determine how RSK's multiple protein partners interact at a molecular level, and how they regulate RSK cellular location, activation and downstream signaling. Additional interaction screens are also proposed to provide new insights into the role RSK plays in Ras-mediated signaling. The third objective of this proposal is to begin an extensive characterization of two RSK targets identified in a yeast two-hybrid screen with the RSK C-terminal kinase domain. The first, FLNa, will provide a molecular understanding of how the Ras-ERK pathway contributes to the regulation of cell spreading and motility. The second, RanBP3, will provide novel information regarding a potential role for RSK in regulating nucleocytoplasmic transport.